Culture medium for insect cells

ABSTRACT

The present invention is directed to a culture medium for insect cells, particularly for culturing insect cells for the expression of proteins using a baculovirus expression system, characterized in that the medium does not contain glutamine.

[0001] This application concerns a culture medium for insect cells.

[0002] The baculovirus expression system using insect cells has becomean important tool for production of recombinant proteins for severalreasons. First, the expression levels are often very high compared tothose obtained in other animal cells, such as CHO-cells. Second,proteins produced using this system are often biologically active due tothe insect cells' ability to perform post-translational modifications,folding and assembly of most proteins. Third, the time from cloning toproduction of the protein is short compared to the time needed toconstruct a stably transformed animal cell line. Fourth, cell deathinevitably follows virus infection of insect cells. This can be anadvantage over other expression systems because it may permit betterexpression of cytotoxic, regulatory or essential cellular genes.

[0003] Foreign proteins are produced during a lytic infection of insectcells with a recombinant virus. Baculoviruses contain a very latehyper-expressed gene, polyhedrin, which is not essential for viralreplication. Placing a gene under control of the polyhedrin promoterallows the production of large quantities of a recombinant protein. Thestrategy for production of a protein involves three distinct stages:

[0004] (a) growing the insect cells from mid to late growth phase;

[0005] (b) infecting the cells with virus containing a gene encoding theprotein of interest; and (c) harvesting and purification of the proteinproduct.

[0006] Insect cells are conventionally cultured in complex mediacontaining inorganic salts and sometimes organic salts, amino acids,sugars, vitamins, trace elements, lipids and protein hydrolysates. Forexample one commercially available medium, called, IPL-41, is availablefrom a number of suppliers. Apart from these components, serum or yeastextract has conventionally been added to provide undefined but essentialnutrients. The amino acid glutamine has hitherto been assumed to beessential for cultured insect cells (Wang, M-Y et al Biotechnol. Prog.1993, 9, 355-361; Kamen, A. A. et al Biotecnol. Bioeng. 1991, 38,619-628;Wang, M-Y et al Biotechnol. Prog. 1993 in press), as it is formost cultured mammalian cell lines.

[0007] We have surprisingly found that insect cells are capable ofsynthesising glutamine themselves. The ability of insect cells to growwithout an external glutamine source has not been described in theliterature before. We have now shown that insect cells are, indeed, ableto grow without glutamine supplied to the medium and that insect cellsare, in fact, capable of growing in a medium without glutamine,glutamate or aspartate.

[0008] According to one aspect of the invention there is provided amedium, for example for culturing insect cells for producing proteins orpolypeptides using a baculovirus expression system, characterized inthat the medium does not contain glutamine.

[0009] The medium may also optionally not contain glutamate and/oraspartate.

[0010] The medium of the invention can be used for growth of the cellsfor the production of recombinant proteins.

[0011] The medium of the invention has several advantages overtraditional, glutamine-containing, insect cell culture media. First,glutamine is generally considered problematic in cell culture because ofits instability. It decomposes spontaneously to pyrrolidone carboxylicacid and ammonia in aqueous solution. This instability is the majorfactor limiting the storage life of a cell culture medium. A mediumwithout glutamine thus results in a markedly longer storage life. Themedium of the invention is also less expensive and simpler to prepare.Further, cells growing in the medium of the invention exhibit a moreefficient metabolism and a decreased secretion of by-products that maybe inhibitory to growth.

[0012] Preferably, the medium comprises an ammonium salt or anotherammonium ion source or at least one amino acid or another compound thatcan be converted to ammonium ions. An external ammonium ion source isthen used as a nitrogen source for biosynthesis of amino acids.Preferably, the concentration of ammonium in the medium is up to 20 mM.However, during certain conditions, as for example, in substrate limitedcultures, the cells may liberate ammonium themselves which in turn canbe used for biosynthesis. Under these conditions addition of ammonium isnot necessary for growth.

[0013] According to another aspect of the invention, there is provided amethod of culturing insect cells, the method comprising growing thecells in a medium according to the invention.

[0014] According to a further aspect of the invention we provide amethod of obtaining a polypeptide including transforming insect cellswith recombinant baculovirus including a gene coding for saidpolypeptide, and growing the transferred cells in a culture medium thatdoes not contain glutamine but which does contain an ammonium ionsource.

[0015] The insect cells may for example be from the fall armyworm,Spodoptera frugiperda.

[0016] The preparation and use of a medium in accordance with theinvention will now be described, by way of example only, with referenceto the accompanying drawings FIGS. 1 to 4 in which:

[0017]FIG. 1 illustrates the growth of insect cells in different media;

[0018]FIG. 2 illustrates the production of alanine and ammonium ions byinsect cells under different conditions;

[0019]FIG. 3 illustrates the growth of insect cells under differentconditions; and

[0020]FIG. 4 illustrates the growth of insect cells in different media.

[0021]FIG. 5 illustrates the expression of the β-galactosidase gene fromcells grown in a medium according to the invention.

EXAMPLE 1

[0022]Spodoptera frugiperda (Sf-9) cells were cultivated in suspensionculture in spinner flasks (Techne, 125 ml) with 50 ml medium, stirred at60 rpm and incubated at 27° C. A new culture was started every secondmonth from ampoules stored in liquid nitrogen. The basic medium used(KBM502) had the following composition in g l⁻¹: glucose, 2.5; KCl,2.87; CaCl₂, 1.0: MgCl₂, 1.07; MgSO₄, 1.36; NaHCO₃, 0.35; NaH₂PO₄, 0.88;choline chloride, 0.02; glutamine 1.0; L-arginine-HCl, 7.0;L-asparagine, 3.5; L-aspartic acid, 3.5; L-glutamic acid, 6.0;L-glycine, 6.5; L-histidine-HCl, 2.0; L-isoleucine, 0.5; L-leucine,0.75; L-lysine-HCl, 6.25; L-methionine, 0.5; L-phenylalanine, 1.5;L-proline, 3.5; L-serine, 5.5; L-threonine, 1.75; L-valine, 1.0;L-cystine-2 HCl, 0.5; L-tryptophan, 1.0; L-tyrosine, 0.72; and, in μgl⁻¹; para-amino benzoic acid, 320; biotin, 160; D-calcium pantothenate,80; folic acid, 80; myo-inositol, 400; niacin, 160; pyridoxine-HCl, 400;riboflavin, 80; thiamine-HCl, 80; vitamin B-12, 240; COCl₂.6 H2O, 50;FeSO₄.7H₂O, 550; MnCl₂O, 20; (NH₄)₆.(Mo₇O₂₄4H₂O), 40; CuCl₂, 158; andZnCl₂, 40. The medium was supplemented with 10% FCS (Gibco), and 50 mg1-1 Gentamicin (Sigma). In the bioreactor experiments, 0.1% PluronicF-68 (Sigma), 40 ppm Anti foam C (Sigma) was added to the medium.

[0023] Glutamine, glutamate and aspartate were omitted from the mediumas indicated below. The concentration of NH₄Cl (when added) was 5 mM.

[0024] Spinner flask cultures

[0025] In the first experiment three media were tested a) KBM502 withoutglutamine, b) KBM502 with 5 mM NH₄Cl but without glutamine, and c)KBM502 with 1 g l⁻¹ glutamine (control culture).

[0026] In the second experiment the following media were tested a)KBM502 with 5 mM NH₄Cl, without glutamine, b) KBM502 with 5 mM NH₄ Cl,without glutamine or glutamate, c) KBM502 with 5 mM NH₄ Cl withoutglutamine, glutamate or aspartate.

[0027] Each medium was tested for growth of cells in triplicate. Spinnerflasks containing 50 ml of medium were inoculated to a cell density of2.5×10⁵ cells/ml⁻¹. The cells were counted once a day.

[0028] Bioreactor cultures

[0029] The medium used for bioreactor cultures was essentially the sameas KBM502, but with the following modifications: it contained TryptoseBroth (Sigma) 2.6 g l⁻¹; the amino acid composition was slightlydifferent (1×Grace's amino acid solution modified for TC 100 (Sigma))and the normal concentration of glutamine was 0.6 g l⁻¹.

[0030] Tank reactors with a working volume of 3.0 (Belach Bioteknik AB,Stockholm, Sweden) equipped with one three blade marine impeller (d=70mm) were used. The stirring speed was 100 rpm and DOT was controlled at30% by intermittent sparging of oxygen at 200 ml min⁻¹. Air was alsopassed above the surface at 100 ml min⁻¹. The pH of the medium wasinitially 6.2 but was not controlled during culture. The culturetemperature was maintained at 27° C. Inoculua were prepared from spinnerflask cultures. The cells were centrifuged and resuspended in freshmedium prior to inoculation. The initial cell density in the bioreactorwas 2×10⁵ cells ml⁻¹.

[0031] In batch cultures the medium was supplemented with 2.5 g l⁻¹glucose and 0.6 g l⁻¹ glutamine. In the glucose fed-batch culture aglucose solution (15 g l⁻¹) was continuously pumped into the reactor(1.8ml h⁻¹) from the time of exhaustion (42 h) of the amount of glucosesupplied by the tryptose broth; and 0.6 g l⁻¹ of glutamine wassupplemented from the beginning of the culture. In the glutamine-limitedculture no glutamine was present in the initial medium but was fed (1.8ml h⁻¹) into the bioreactor using a glutamine solution (3 g l⁻¹) fromthe beginning of the experiment. Glucose (2.5 g l⁻¹ was added from thebeginning. The combined glucose and glutamine fed-batch culture was fedinitially with only a glutamine solution but when the initial amount ofglucose was exhausted (44.5 h), a mixed solution of glucose (15 g l⁻¹)and glutamine (3 g l⁻¹) was fed at a rate of 1.8 ml h⁻¹.

[0032] Sampling and analyses

[0033] Cell counting was performed using a Büker counting chamber andcell viability was determined by trypan blue exclusion. Samples forammonium, lactate, glucose, amino acid and other organic aciddeterminations were centrifuged and stored frozen until analyzed. Theammonium ion concentration was measured with an ammonium selectiveelectrode (Orion). Lactate and uric acid were analyzed enzymaticallyusing test kits from Sigma while citrate and succinate were determinedby test kits from Boehringer Mannheim. Glucose was analyzed with an YS1glucose analyzer. Analyses of amino acids were performed on a BiotronikLC 5001 amino acid analyzer using ninhydrin as detection reagent.

[0034] Batch and Fed bath bioreactor cultures

[0035] The growth of Sf-9 cells and the concentration of glucose andglutamine in a) a batch; b) a glutamine-limited fed batch c) aglucose-limited fed batch; and d) a dual glucose and glutamine-limitedfed batch culture is shown in FIG. 1. Symbols: viable cells, filledsquares; glucose, open squares; glutamine, open circles.

[0036]FIG. 2 illustrates the production of alanine and ammonium ions bySf-9 cells in a) batch culture, b) glutamine-limited fed batch culture,c) glucose-limited fed batch culture, and d) dual glucose- andglutamine-limited fed batch culture. Symbols: Alanine —∘— and ammonium—— formation by Sf-9 cells.

[0037] In the batch culture the formation of alanine was considerable—atypical metabolic response of insect cell cultures (FIG. 2a). Theconcentration of alanine increased from 3 mM at the start to almost 8mM. On the contrary, the formation of ammonium ions was insignificant.

[0038] In the glucose-limited fed batch culture, the glucose feeding wasstarted at 42 h when the initial quantity of glucose, (unfortunatelysupplied by the tryptose broth) was consumed (FIG. 1c). After that theglucose concentration remained very low (below 0.2 mM) throughout theculture. The formation of alanine is not only completely depressed, butalanine is actually consumed after glucose limitation was established,while, at the same time, ammonium production is triggered (FIG. 2c). Theconcentration of ammonium ions increases abruptly from 0.5 mM to around4 mM. Ammonium formation continues, although at a lower rate, afterdepletion of glutamine (150 h), indicating that it is released also fromother amino acids.

[0039] In the glutamine-limited culture, glutamine was fed at a constantrate from the beginning resulting in a small increase in concentrationto a maximum of 0.5 mM whereafter it declined below the detection limit(FIG. 1b). In this type of culture alanine production takes place (FIG.2b) but the accumulation is less than that in the batch culture. Noincrease in ammonium ion concentration is observed.

[0040] Finally, in the glucose and glutamine-limited culture, whereglutamine and glucose were fed simultaneously, low concentrations ofboth substrates were established (FIG. 1d). Very little alanine orammonium was formed in this culture (FIG. 2d).

[0041] During glutamine limitation, the consumption of glutamine wasmuch less than that of any other amino acid as shown in Table 1.

[0042] This small amount seemed insufficient for the biosynthetic needof the cells. Therefore it would appear that glutamine biosynthesisoccurs in the glutamine-limited cultures. The increase in theconcentration of glutamine in the stationary phase of the batch culture,after exhaustion of the initial amount (FIG. 1a), also supports the ideathat Sf-9 cells can synthesise glutamine. From Table 1 it is alsoevident that the consumption of glutamate and aspartate increased in thefed batch cultures.

[0043] Growth of insect cells in medium without glutamine FIG. 3illustrates the growth of cells cultivated in media with or withoutglutamine and with or without 5 mM NH₄Cl Cells cultivated in a mediumwithout glutamine but supplemented with NH₄Cl, reached almost the samemaximum cell density, 2.9×10⁶ cells ml⁻¹, as cells cultivated in astandard medium containing glutamine, although the growth rate issomewhat lower (FIG. 3). This may be explained by the extra burden thatthe synthesis of glutamine exerts on the cells. Cells grown in a mediumlacking both glutamine and NH₄Cl grow very poorly; the maximum celldensity does not exceed 6×10⁴ cells/ml (FIG. 3). This experiment hasbeen repeated many times, and the cell culture has now been maintainedin a medium lacking glutamine for several months.

[0044] This experiment clearly demonstrates that Sf-9 cells are capableof growing well without an external source of glutamine, provided thatammonium ions are supplied to the medium. To add an ammonium salt to amedium for animal cells has generally been regarded as veryunfavourable, as external ammonium is inhibitory for most culturedanimal cells, even at rather low levels. We cannot see any negativeeffects of added NH₄Cl below a concentration of 20 mM.

[0045] Biosynthesis of glutamine, by glutamine synthetase, requires thesubstrates glutamate and ammonium. We have shown earlier that noammonium was formed during growth of Sf-9 cells in a batch culture withexcess glucose (FIG. 2a) and in a glutamine limited culture with excessglucose (FIG. 2b). Thus, external ammonium has to be supplied as anitrogen source for the biosynthesis of glutamine. However, duringglucose limitation Sf-9 cells excreted ammonium into the medium (FIG.2c). In such and similar cases it may not be necessary to add ammoniumto the medium.

[0046] Growth of insect cells in medium without glutamine, glutamate andaspartate.

[0047]FIG. 4 illustrates the growth of insect cells in medium withoutglutamine, glutamate and aspartate. FIG. 4 clearly shows that Sf-9 cellsare able to grow without external sources of glutamine, glutamate,glutamate and aspartate, provided that ammonium is supplied to themedium. No significant difference can be seen in the growth between thethree media.

[0048] These results indicate further that Sf-9 cells do indeedsynthesize glutamine, glutamate and aspartate because all three aminoacids are needed for various biosynthetic functions and for proteinsynthesis. It is likely that glucose and ammonium ions are the ultimatesubstrates for the synthesis of the three amino acids. In this respect,the metabolism of Sf-9 cells resembles that of many microorganisms, butit is a very uncommon type of metabolism in cultured animal cells.

[0049] Further experimentation has confirmed that Sf-21 cells arecapable of growing in a medium containing no glutamine but to which 5 mM NH₄Cl has been added.

EXAMPLE 2

[0050] Expression of β-galactosidase in a glutamine-free medium To studythe effect of glutamine-free conditions on recombinant proteinproduction, Sf-9 cells were infected with a recombinant baculovirusduring growth in the glutamine-free but ammonium supplemented medium (5mM NH₄Cl) and the result compared to that from standard conditions (i.e.a medium with glutamine but without added NH₄Cl). The yield ofβ-galactosidase (24-72 h after infection, FIG. 5) indicates that theproduction of the recombinant protein is not negatively affected by theglutamine-free conditions. TABLE Consumption of amino acids in batch andfed batch (FB) cultures of insect cells. Consumption (μmol/10⁶ cells)¹Glucose and Glucose Fed Glutamine Glutamine Amino acid Batch Batch FedBatch Fed Batch Glutamine 1.85 1.85 0.04 0.02 Glutamate 0.11 0.29 0.450.62 Aspartate 0.01 0.03 0.34 0.20 Asparagine 0.09 0.15 0.28 0.26 Serine0.46 0.33 0.76 0.70 Lysine 0.13 0.22 0.37 0.40 Threonine 0.11 0.13 0.210.21 Arginine 0.23 0.27 0.40 0.43 Valine 0.14 0.11 0.20 0.27 Isoleucine0.16 0.10 0.17 0.20 Leucine 0.29 0.25 0.36 0.36 Tyrosine 0.12 0.10 0.160.12 Phenylalanine 0.12 0.10 0.15 0.17 Tryptophan 0.04 0.07 0.08 0.07Methionine 0.15 0.15 0.18 0.15

[0051]

1. An insect cell culture medium characterised in that the medium doesnot contain glutamine.
 2. An insect cell culture medium according toclaim 1 which also does not contain glutamate.
 3. An insect cell culturemedium according to claim 1 or 2 which does not contain aspartate.
 4. Aninsect cell culture medium according to any preceding claim whichcontains an ammonium ion source.
 5. A method of culturing insect cellscomprising growing the cells in a medium according to any precedingclaim.
 6. A method of obtaining a polypeptide including transforminginsect cells with recombinant baculovirus including a gene coding forsaid polypeptide, and growing the transformed cells in a culture mediumthat does not contain glutamine but which does contain an ammonium ionsource.
 7. A method according to claim 5 or claim 6 wherein the cellsare from Spodoptera frugiperda.
 8. A method according to claim 7 whereinthe cells are from the cell lines Sf-9 or Sf-21.